P18IIIB is a highly immunogenic peptide from the V3 loop of the HIV-1 gp160 envelope protein that is presented promiscuously by multiple class I MHC molecules. Understanding the molecular basis for promiscuous presentation may have many practical applications. As the highly prevalent HLA-A2.1 class I molecule is known to present P18IIIB for recognition by cytotoxic T lymphocytes (CTL) found in peripheral blood mononuclear cells of HIV+ donors, a P18IIIB-specific CTL line was generated from an HLA-A2.1 +, HIV- donor in order to define the molecular basis for, and ultimately improve upon the binding of, this peptide to HLA-A2.1. The minimal epitope recognized by the line was a decamer, I10, with the sequence RGPGRAFVTI. Many of the agretopic and epitopic residues identified were identical to those involved in the corresponding interactions of I10 with the H-2Dd MHC molecule and murine CTL. The I10 peptide does not contain the described HLA-A2.1 binding motif. Instead Pro at P3, a Phe at P7 and an Ile at P10 are utilized for MHC binding. Agretopic residue similarities with the hepatitis B nucleocapsid decamer suggest that these residues may comprise an alternative motif of anchors utilized by decamers for binding to HLA-A2.1. Sequence analyses of endogenous peptides bound to HLA-B44 revealed two potential dominant anchor residues: Glu at P2 and Tyr, or occasionally Phe, at P9. Using this information, we identified antigenic peptides from the influenza virus components nonstructural protein 1 and nucleoprotein that are presented by HLA-B44 to antiinfluenza type A cytotoxic T lymphocytes. In addition, cytotoxic T lymphocytes induced by these antigenic peptides were shown to be capable of recognizing endogenously processed peptides from influenza-infected cells, indicating a potential use for these peptides in vaccine development. It is possible to predict the binding stability of peptides for class I molecules by applying coefficients, deduced from a large amount of binding data that quantify the relative contribution of amino acids at each position in the peptide. We described the molecular basis for these coefficients, and demonstrate that estimates of binding stability based on the coefficients is generally concordant with experimental measurements of binding affinities. Apparently, peptide binding affinity is largely controlled by the rate of dissociation of the HLA/peptide/beta2m complex, whereas the rate of formation of the complex has less impact on peptide affinity. Although peptides that bind tightly to class I molecules, including many antigenic peptides, can be readily identified using the coefficients, a few antigenic peptides bind much more weakly and can be overlooked by this approach.